Cocaine is an ecgonine ester compound of the formula: ##STR1## (ref. 1--a list of references appears at the end of the descriptive text. This paper provides an overview of nomenclature. Compound names used in this specification are defined in this article).
The abuse of cocaine represents a major threat to the social and economic fabric of many developed countries. Although several dopaminergic agents and the tricyclic antidepressant desipramine have been clinically tested, effective therapies to assist drug-addicted individuals in their return to drug-free life still are not available. Mobilizing the immune system to "block" drugs from reaching their sites of action in the central nervous system represents a potential, but as yet poorly explored, means of therapeutic intervention.
It is well known that drugs of abuse can be rendered inactive by disrupting a structural feature either required for the interaction with their respective receptors or necessary for transport. Thus, in cocaine, the presence of the benzoyl ester moiety in the molecule is essential for maintaining its activity. Therefore, if antibodies possessing cocaine-specific esterase activity could be induced, such catalytic antibodies could potentially act in vivo to neutralize the pharmacological effects of the drug in an immunized individual. Enzymes and abzymes (otherwise known as catalytic antibodies) apparently employ a similar mechanism for the catalysis of hydrolysis.
Abzymes, as any catalyst, lower the energy required to proceed through the transition state between the starting compound and the respective reaction products. Thus, a catalytic antibody binds to and stabilizes a shape corresponding to the transition state with little or no energy expenditure on the part of the substrate.
Depending on the presence of other factors, the substrate then could proceed to the product or to return to its starting form. In the case of hydrolysis, water must be present, since the hydroxyl group of the water, due to its nucleophilic properties, enters the protransition state and forms the proper transition state for the hydrolysis, and the hydrolysis then takes place. Therefore, a catalytic antibody should be ideally made against such a transition state. However, since transition states are unstable by definition, antibodies have to be made against stable molecules which structurally mimic the transition state (transition state analogs). It has been established that the transition state (ref. 2) for carboxylate ester hydrolysis is centered around unstable formally "pentavalent" carbon, and consequently it can be mimicked by a stable phosphonate ester (ref. 3) since phosphorus is stable pentavalent and shapes and charge distribution of both resemble each other fairly closely. However, esters are among the most common functional groups in living organisms, and thus it is essential that the abzyme is devoid of any general esterase activity and is endowed with very specific benzoyl esterase activity in the context of the cocaine molecule. To achieve this objective, it is crucial that the transition state analog does not disrupt structural features defining specificity of interaction between cocaine and the recognition moiety of the abzyme. If this condition is not met, the antibodies made against such transition state analogs will not be sufficiently specific to be practical.
It is recognized that polar groups in a molecule tend to be the focal point of B-cell (i.e. antibody reactive) epitopes. In cocaine, there are three polar groups, namely the bridgehead nitrogen (methylated), the methyl ester, and the benzoyl ester. As explained above, since the benzoyl ester is the target for the hydrolysis by a catalytic antibody, the transition state for the hydrolysis of the benzoyl ester can be mimicked by substituting phenylphosphonate for benzoate in the cocaine molecule. Such a phosphonate has to be linked to a carrier protein, as is conventionally required to enhance the immunogenicity of small molecules. Linkers have to be of appropriate length to maintain the transition state analog at the optimal distance from the antibody binding site. If the linker is too short, the carrier protein could interfere sterically, while, if it is too long, the linker may fold back to the protein, so that the transition state analog would adhere to the protein molecule or its fragments after processing.
Four sites for anchoring the linker on the cocaine molecule are identifiable (listed in order of increasing synthetic difficulty):
(i) a substitution of the N-methyl group by an alkyl chain, the other end of which is bound to a carrier protein (e.g. utilizing the amino group of a lysine in the carrier protein); PA1 (ii) a substitution of the methyl ester by a bifunctional molecule, such as a dicarboxylic acid, the other end of which again is bound to a carrier protein, either directly or through an extension chain; PA1 (iii) p-substitution at the phenyl ring of the phenylphosphonate group with a chain linked again to a carrier protein directly or through an extension chain; and PA1 (iv) a substitution of a ring hydrogen in the ecgonine ring system by a chain of carbon atoms, the other end of which is functionalized so that a bond to a carrier protein can be formed. PA1 (b) the group (-Y-functional group), wherein Y is a linker group, including an alkylene radical, and PA1 (c) the group (-Y-carrier molecule), wherein Y is a linker group, including an alkylene radical.
Although the third choice (iii) appears to be the best one since it disturbs least of all the important recognition elements of cocaine and remains still within the reach of organic synthetic methodology for a possible future mass production, an attempt was described to link a phenylphosphonate analog of cocaine (ref. 4) via an alkyl chain originating in the nitrogen function utilizing anchoring site (i). Although a number of binding monoclonal antibodies have been isolated, none of them was endowed with the desired catalytic activity, thus confirming the conclusion of the discussion hereinabove.
At least two attempts have been made utilizing the anchoring site (ii). The transition state analog using a specific linker (ref. 5) was described that using the state of the art methodology made possible isolation of two catalytic monoclonal antibodies with small, albeit detectable catalytic activity. Identical transition state analogs using a different linker to BSA or KLH (compounds 5a, 5b, FIG. 1--ref. 6) gave a polyclonal binding antibody in rabbits, and several binding monoclonal antibodies, none of them endowed with catalytic activity. This result could be expected, as it has been outlined hereinabove.